Hepatitis C Virus (HCV) infects over 170 million persons worldwide. It is the leading cause of liver disease in the U.S. and is responsible for most liver transplants. Current treatments for this infectious disease are inadequate; therefore new therapies must be developed. We as well as others have obtained evidence for a multi-protein complex that involves all of the known nonstructural (NS) proteins encoded by the virus. NS3, NS4A, NS4B, NS5A, and NS5B appear to interact structurally and functionally. We will apply state-of-the art biochemical approaches for studying the mechanism of RNA unwinding, translocation, and RNA binding of NS3-4A, which is believed to be the biologically relevant form of this enzyme. We will examine potential NS3-4A interactions with other NS proteins in biologically relevant HCV replicons. We have found that the NS3 helicase stimulates the activity of the NS5B polymerase dramatically. The mechanism for this functional interaction will be investigated. We have recently solved the crystal structure of an apparent dimeric form of NS3 helicase domain in which two molecules of the enzyme are bound to the same strand of nucleic acid. We wilt test the biological and biochemical significance of the interactions revealed by this new structure. Our work has identified protein surfaces of NS3 that are required for replication of the HCV genome, but are not part of known helicase domains. These surfaces are likely to be involved in highly specific, protein-protein interactions and are therefore targets for disruption of multi-protein complexes that are responsible for HCV replication. Sites for interaction between NS3 and other HCV proteins will be determined by chemical crosslinking coupled with mass spectrometry. We have found that NS5A binds tightly to RNA and to NS5B. We will now examine possible functional interactions between NS5A, NS3, and NS5B and attempt to solve the crystal structure of NS5A.